The present invention relates to the characterization of an antigenic site belonging to an immuno-dominant multiepitope region located in the N-terminal end of the Core or nucleocapsid protein (or p21 protein) of the hepatitis C virus (HCV), [B. Hosein et al., Proc. Natl. Acad. Sci. USA, 88, 3647-51 (1991)] as well as to the applications resulting therefrom, in particular in the diagnosis, treatment or prevention of an HCV infection.
In accordance with the document EP-A-0 569 309, in the name of the Applicant, a peptide is known which extends from the amino acid at position 2 to the amino acid at position 45 of the N-terminal end of the HCV nucleocapsid protein, and whose sequence is identified by SEQ ID NO: 1. This peptide determines an immunodominant region which is sufficient, on its own, for obtaining the same sensitivity and specificity, in terms of detection of antibodies directed against HCV, as with the nucleocapsid protein in its entirety.
Continuing its work on the peptide defined above, the Applicant has discovered that:
the region extending from amino acid 15 to amino acid 39 of the N-terminal end of the nucleocapsid protein defines the location of an immunodominant epitope,
this immunodominant epitope is of conformational type, and corresponds to a three-dimensional unit of helix-loop or elbow-helix type, in which the two helices are arranged substantially perpendicularly to each other.
Thus, the invention relates to a structural peptide, recognized by the antibodies directed against a polypeptide comprising the sequence of amino acids 2-45 of the N-terminal end of the Core protein (p21) of the hepatitis C virus (HCV), which comprises, or consists of, a tertiary structure consisting of at least a first peptide fragment having a secondary structure in the form of an xcex1 helix, a second peptide fragment having a secondary structure in the form of an a helix and a third joining peptide fragment linking the two a helices, these two xcex1 helices being substantially perpendicular to each other in space. The angle formed by the two xcex1 helices is advantageously 90xc2x0xc2x110xc2x0.
As used herein, the phrase xe2x80x9chepatitis C virusxe2x80x9d includes all of the naturally occurring forms of hepatitis C virus.
The third joining peptide fragment is preferably chosen from loops and elbow-shaped secondary structures, in particular xcex2 elbows.
According to the present invention, are excluded from the preceding definition, and from the following definitions, all protein or peptide compounds comprising or consisting of the N-terminal end of the p21 protein, this end being considered as starting at the amino acids at position 1 or the amino acid at position 2. The excluded portion preferably includes at least the first 5 amino acids of the N-terminal end of the p21 protein (the first 4 amino acids of the N-terminal end of SEQ ID NO:1); more preferably at least the first 8 amino acids of the N-terminal end of the p21 protein (the first 7 amino acids of the N-terminal end of SEQ ID NO:1); even more preferably at least the first 11 amino acids of the N-terminal end of the p21 protein (the first 10 amino acids of SEQ ID NO:1); and most preferably the first 14 amino acids of the N-terminal end of the p21 protein (the first 13 amino acids of the N-terminal end of SEQ ID NO:1).
Advantageously, the peptide sequence of the peptide of the invention is chosen from any peptide sequence equivalent to SEQ ID NO: 2, such as SEQ ID NO: 3 to 10.
Preferably, the peptide contains fewer than 45 amino acids. More preferably, the peptide contains no more than 35 amino acids. Even more preferably the peptide contains no more than 30 amino acids. Most preferably, the peptide contains about 25 amino acids.
Advantageously, the peptide of the invention is different from the sequence SEQ ID NO: 11 (Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Val Val Gly Gly Val Tyr Leu Leu Pro Arg).
Preferably, the peptide of the invention is different from fragment 4-39 of SEQ ID NO:1.
Before describing the present invention in greater detail and presenting the importance and the applications thereof, a definition of terms used in the description and the claims is given below.
Conformational epitope is understood to mean a protein region recognized by antibodies and determined in particular by amino acids which may be distant in the protein sequence but which, because of the folding of the polypeptide chain, become close to each other in space and may thus form a unit capable of being recognized by the antibody.
Peptide designates a stretch of amino acids which is obtained by chemical synthesis or by genetic recombination techniques. The peptides according to the invention may be obtained by conventional synthesis methods, for example with an automated peptide synthesizer, or by genetic engineering techniques comprising the insertion of a DNA sequence encoding said peptide into an expression vector such as a plasmid or a virus, and the transformation of eukaryotic or prokaryotic cells with this expression vector and culturing of these cells.
Peptide sequence equivalent to a reference peptide sequence (such as SEQ ID NO: 2 in the case of the present invention), said reference peptide sequence having a reference unit, is understood to mean an amino acid sequence modified by insertion and/or deletion and/or substitution and/or extension and/or shortening and/or chemical modification of one or more amino acids, as long as these modifications, on the one hand, substantially preserve, or even amplify, the antigenic properties of said reference peptide sequence and, on the other hand, essentially conserve the reference tertiary structure of said reference peptide sequence.
Thus, xe2x80x9cequivalent sequencexe2x80x9d is understood to mean in particular the sequences in which one or more amino acids are substituted by one or more other amino acids; the sequences in which one or more amino acids of the L series are replaced by one or more amino acids of the D series; the sequences into which there has been introduced a modification of the amino acid side chains, such as an acetylation of the amine functions, a carboxylation of the thiol functions, an esterification of the carboxyl functions, a substitution of an oxygen atom by a sulfur atom, and the like; a modification of the peptide bonds, such as carba, retro, inverse, retro-inverse, reduced and methyleneoxy bonds.
Peptide sequences equivalent to SEQ ID NO: 2 also comprise those of mimotope peptides which have a very different peptide sequence from SEQ ID NO: 2, while having the same antigenic properties and potentially the same tertiary structure as SEQ ID NO: 2.
Peptide sequences equivalent to SEQ ID NO: 2 also comprise those of mimotope peptides which have a peptide sequence little different from SEQ ID NO: 2, while having the same tertiary structure and the same antigenic properties as SEQ ID NO: 2. These mimotope peptides are described as an example by the peptide sequences SEQ ID NO: 3 to 10.
The secondary structure of a peptide fragment is defined as a three-dimensional structure essentially stabilized by hydrogen bonds involving the atoms which form the primary structure. Several types of secondary structures, and in particular the structure in the form of an a helix and the structure in the form of a xcex2 elbow, can be distinguished.
The xcex1 helix has been described by Pauling et al. [L. Pauling, Proc. Natl. Acad. Sci. USA, 37, 205-211 (1951)], and corresponds to a right-handed helix. The xcex1 helix is essentially stabilized by hydrogen bonds between the oxygen of the carbonyl group of the amino acid at position i and the hydrogen of the amide group of the amino acid at position i+4. These hydrogen bonds are almost parallel to the longitudinal axis of the helix, and the N, H and O involved in the hydrogen bond are practically colinear. The neighboring amino acid residues are 1.5 xc3x85 apart on the axis of the a helix, and a complete turn corresponds to 3.6 amino acid residues. The movement along the axis of the helix is therefore 5.4 xc3x85 per turn.
The parameters of the xcex1-helix structure are the following:
Angles in degrees:
xcfx86 xe2x88x9257
"psgr" xe2x88x9247
xcfx89 180
Amino acids per turn 3.6,
Interresidue distance in xc3x85(Cxcex1i-Cxcex1+1) 1.5
The xcex2 elbows are considered as a secondary structure of which at least 7 different types have been described [P. Chou and G. D. Fasman, J. Mol. Biol., 115, 135-175 (1977)]. They allow the folding of the polypeptide chain and involve a minimum of 4 amino acids. They generally allow a change of direction between 2 secondary structures.
Finally, the loops, which are not generally considered as a secondary structure, correspond to a continuous segment of polypeptide chain most often describing the letter xcexa9 in space, hence their name of omega loop [J. Leszczinski and J. Rose, Sciences, 234, 849-855 (1986)]. A loop is defined by its length and a short distance between its ends. The loops are stabilized and defined by interactions at the level of the side chains of their constituent residues.
To obtain a unit, or reference tertiary structure corresponding to the preceding definition according to the invention, it preferably comprises a primary structure equivalent to the amino acids extending from the amino acid at position 15 to the amino acid at position 39 of the N-terminal end of the nucleocapsid protein of the hepatitis C virus.
More advantageously, this primary structure comprises, or consists of, any peptide sequence equivalent to SEQ ID NO: 2. When the structural peptide has the primary structure identified by SEQ ID NO: 2, the first peptide fragment, having a secondary structure in the form of an xcex1 helix, consists of the sequence Thr Asn Arg Arg Pro Gln Asp Val Lys Phe (1 to 10), the second peptide fragment, having a secondary structure in the form of an a helix, consists of the sequence Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg (15 to 25), and the third peptide fragment consists of the sequence Pro Gly Gly Gly (11 to 14).
The invention also relates to an antigenic compound, recognized by the antibodies directed against a polypeptide comprising, or consisting of, the sequence of amino acids 2-45 of the N-terminal end of the Core protein (p21) of the hepatitis C virus (HCV), as well as to an immunogenic compound capable of inducing the production of antibodies, said antigenic or immunogenic compound comprising a peptide of the invention, excluding any protein or peptide compound comprising or consisting of the N-terminal end of the p21 protein, this end being considered as starting at the amino acid at position 1 or the amino acid at position 2.
By way of examples, the peptide according to the invention may be conjugated, according to techniques well known to the person skilled in the art, with a carrier molecule, such as, for example, a natural or recombinant protein, a synthetic polymer of amino acids or with aliphatic chains, a nucleic fragment or a tracer or marker molecule, such as, for example, an oligonucleotide, an enzyme, such as horseradish peroxidase, alkaline phosphatase or xcex2-galactosidase or alternatively a radioelement. The peptide of the invention may be attached to any support.
The monoclonal or polyclonal antibodies capable of being obtained, for example, by immunization of an animal or immunization in vitro with the immunogenic compound defined above, under conventional physicochemical conditions, also constitute a subject of the present invention.
The subjects of the invention which are described above, namely antigenic or immunogenic compound and peptide are capable of being used for detecting and/or quantifying antibodies directed against the p21 of HCV, and the abovementioned antibodies may be used for detecting and/or quantifying the antigens of HCV.
These subjects may, in addition, be applied to the preparation of a diagnostic, prophylactic or therapeutic composition intended for detecting, preventing or treating an infection with HCV.
Accordingly, the invention also relates to a pharmaceutical composition comprising a unit, a peptide, a compound, and/or antibodies defined above.
The invention relates, in addition, to:
a method of detecting and/or quantifying, in a sample, either antibodies directed against the HCV nucleocapsid protein, or antigens of HCV, comprising the steps consisting in bringing said sample into contact with, either a peptide and/or a compound defined above, or antibodies of the invention, and in detecting the formation of an immune complex, either between said antibodies and said peptide or compound, or between said antigens and said antibodies of the invention;
a method of detecting and/or quantifying, in a sample, all or part of the HCV RNA, comprising the steps consisting in bringing said sample into contact with a peptide, and/or a compound of the invention, and in detecting the formation of a complex between said RNA and said peptide or compound.
The invention relates, in addition, to a complex comprising a peptide of the invention, and a molecular structure specifically bound to said peptide, and which may, for example, be selected from peptide fragments, nucleotide fragments and chemical molecules such as those of the functionalized aromatic type. The complexing of a peptide of the invention which exhibits a high affinity with the viral RNA in particular, can prevent the attachment of this RNA and the expression of the latter. This complex therefore has application in the treatment of an HCV infection.